(A) Mice received a single IP injection of BrdU (1 mg) and were humanely killed 24 h later or received BrdU for 3 d in their drinking water (0.8 mg/ml) and were humanely killed at the end of the labeling period. Lymph node cells were collected and stained with anti-Foxp3, anti-CD4, anti-CD44, anti-Ki-67, and anti-BrdU. Numbers inside the quadrants are the mean frequencies of BrdU⁺/Ki-67⁺ and BrdU⁻/Ki-67⁺ cells (mean ± standard deviation [SD] for three replicate animals). Numbers with arrows are the MFI of Ki-67 staining (mean ± SD). (B) B6 mice were infected IP with 2 × 10⁵ plaque-forming unit (PFU) of LCMV Armstrong; 60 d later, spleen cells were stained with an I-A^b-GP66-77 (DIYKGVYQFKSV) tetramer, anti-CD44, anti-CD4, and anti-Ki-67. In the upper panel, the mean frequency of CD44^bright cells among the tetramer+ and tetramer− cells from three replicates is shown (mean ± SD). The proportion of Ki-67⁺ cells among the CD44^bright tetramer− and tetramer+ cells is shown in the lower panel. (C) B6 mice infected with LCMV Armstrong 15 d earlier were analyzed for CD44 expression and tetramer binding. The lower panels represent the proportion of Ki-67⁺ cells among CD44^bright tetramer+ and tetramer− cells. (D) Normal B6 mice were placed on BrdU (0.8 mg/ml) in their drinking water for a period of 10 d. On days 1, 3, 5, and 10, mice were humanely killed and lymph nodes cells were collected and stained with anti-Foxp3, anti-CD4, anti-CD44, and anti-BrdU.

2×10⁶ CD45.1 OT-II CD4 T cells were injected IP into CD45.2 FcγRγ^−/− B6 mice. 24 h later, mice were treated with Y3P (1.8 mg IP) or mouse immunoglobulin G (IgG), and 1 d later they were immunized IP with ovalbumin peptide (10 µg) plus LPS (25 µg) or LPS only. BrdU was given in drinking water from the time of immunization; 3 d later, lymph node cells were collected and stained with anti-CD45.1, anti-CD45.2, anti-CD44, anti-BrdU, and anti-Ki67. Contour plots were used because of the low number of cells available for analysis from mice treated with Y3P. (B) Numbers of CD45.1 and CD45.2 and of Ki-67⁺ BrdU⁺ cells from immunized control or Y3P-treated mice. (C) FcγRγ^−/− B6 mice were treated for 3 d with 1.8 mg of Y3P followed by a 6-h BrdU pulse (1 mg). Numbers in the quadrants represent the frequency of BrdU-positive and -negative and Ki-67-positive and -negative cells for an individual animal. Lower panels present means and SDs for numbers of BrdU⁺ and Ki-67⁺ cells among the three animals in each group.

(A) 1 million CFSE-labeled MP CD4 T cells were transferred into Rag 2−/− mice that received a single dose of 0.5 mg of anti–IL-7, anti–IL-15, or 1.8 mg of Y3P or were untreated. On day 3, the mice received a second dose of anticytokine antibody or of Y3P. Mice were humanely killed on day 6 and single cell suspensions from lymph nodes were stained by anti-CD4 followed by flow cytometric analysis of CFSE dilution. Numbers inside the histograms represent the frequency of cells that have divided at least once (mean ± SD, from three mice per group). (B) 1 million CFSE-labeled MP CD4 T cells were transferred into Rag2−/− recipients. On day 3, animals were humanely killed and 50×10³ CFSE low and high cells were sorted, followed by PCR amplification using Vβ2 and Jβ1.1 primers (see Figure 6A). CDR3 sequences from the amplified DNA were plotted according to the frequency with which they appeared among the 42 sequences obtained from CFSE low and high Vβ2/Jβ1.1 cells, respectively.

Sequence diversity within Vβ2-Jβ1.1 and Vβ4-Jβ1.1 Ki-67^bright and Ki-67^negative CD44⁺ CD4 T-cell populations. Shown is the percentage occurrence of a given sequence in two mice. N is indicated above each table for Ki-67^bright and Ki-67^negative cells. NUS indicates number of unique sequences that occur only once in either the Ki-67^bright and Ki-67^negative cells. Each table represents the numbers of sequences that occur once, twice, three times, four times, five times, and more than five times among the Ki-67^bright and Ki-67^negative populations.

(A) Conventional B6 and B6 GF mice received an IP injection of 1 mg BrdU and were humanely killed 6 h later. Lymph nodes cells were collected and stained with anti-Foxp3, anti-CD4, anti-CD44, anti-BrdU, and anti-Ki-67. The numbers in the upper quadrants represent the mean percent of BrdU⁺ and Ki-67⁺ cells (mean ± SD for three replicate animals). (B and C) Numbers of CD4⁺, CD44⁺, CD44⁺ Foxp3⁻, Ki-67⁺, and BrdU⁺ cells in peripheral LN (B) and mesenteric LN (C) of conventional (black bars) and GF (white bars) mice.

Normal B6 mice were either untreated or received two doses of 0.5 mg of anti–IL-15, anti–IL-7Rα, or anti–IL-2 antibody spaced 3 d apart. Mice were humanely killed on day 7 and lymph node cell suspensions were stained with anti-CD4, anti-CD44, and anti–Ki-67. *p<0.05.

Experimental approach to analyze the CDR3 sequences of Vβ2-Jβ1.1 and Vβ4-Jβ1.1 CD4⁺ CD44⁺ Foxp3⁻, KI-67^bright and Ki-67^negative cells from individual mice. Cells were FACS sorted into lysate buffer containing proteinase K. One-third of the lysate was subjected to PCR with specific Vβ and Jβ1.1 primers (see Methods). PCR products were cloned into the Topo vector followed by bacterial transformation and single colony isolation. PCR with universal M13 primers were used on each colony to amplify the Vβ2-Jβ1.1 and Vβ4-Jβ1.1 gene segments followed by sequencing with the universal T3 primer.

Sequence diversity among Ki-67^bright and Ki-67^negative Vβ2-Jβ1 CD44⁺ CD4 T-cell populations. Shown is the percentage of times an individual sequence occurred in two GF mice.